Science - USA (2020-10-02)

C-terminal mutations on anSry-T–deficient
background confirmed that absence of the S18
degron is strictly required for SRY-S to activate
the male pathway in vivo (fig. S8B and table S1).
Thus, we conclude that the C-terminal degron
prevents endogenously expressed SRY-S pro-
tein from accumulating to a level required for
activating the male pathway, not only inSry-
T–deficient mice but also in WT XY mice.
Several previous studies showed thatSry
transgenes encoding only SRY-S can induce

testis development in XX mice under experi-

mental conditions ( 1 , 8 , 9 , 13 ). The sex reversal

occurred in 25% ( 1 )to75%( 8 )ofmiceandwas

shown to correlate with transgene expression

levels ( 9 ), which was in turn influenced by in-

tegration site and/or copy number ( 14 ). In sup-

port of this concept, heterozygousNr5a1-Cre;

Sry-S-R26 knock-in XX mice did not develop

as males but homozygotes did (Fig. 3D). There-

fore,Sry-Stransgene expression above a cer-

tain threshold can overcome the destabilizing

effects of the degron. However, under physio-

logical conditions, it is not SRY-S, but rather

degron-free SRY-T, that is the bona fide testis-

determining factor in the mouse.

Sryevolved from the single-exon geneSox3

( 15 , 16 ), and all previously characterized euthe-

rianSrygenes are single exon. Therefore, our

findings raise the important questions of how

and why an additional exon arose in mice. We

found that DNA sequences similar to the mouse

Sry-Sdegron are conserved inSryfrom other

SCIENCEsciencemag.org 2 OCTOBER 2020•VOL 370 ISSUE 6512 123

Fig. 3. SRY-T is the predominant SRY isoform
and is sufficient to sex-reverse XX mice.
(A) Establishment of mouse lines carrying
Sryknocked-inRosa26alleles namedSry-T-R26KI
andSry-S-R26KI. Cre/loxP-mediated genome
recombination inducesSryexpression by removing
the triple-poly(A) stop cassette between the
promoter and theSryCDS. (B) Relative mRNA
expression ofSryin E11.5 gonads of the indicated
genotypes.Sryexpression was normalized to
Gapdh. Data are shown as means ± SD. ns, not
significant, Mann–WhitneyUtest. (C) Capillary-based
immunodetection of E11.5 gonadal cell lysates
of the indicated genotypes. Expression levels of FLAG
relative tob-ACTIN are indicated. (D) External
phenotypes of 2-month-old mice of the indicated
genotypes.Nr5a1-Cre;Sry-T-R26KI/+XX mice (n= 6)
developed as males butNr5a1-Cre;Sry-S-R26KI/+
XX mice (n= 10) did not.Nr5a1-Cre;Sry-S-R26KI/KI
XX mice (n= 3) developed as males. (E) Coimmu-
nostaining profiles of SRY and NR5A1 in E11.5
gonads of the indicated genotypes. Subcellular
localization of SRY-T and SRY-S are shown; for more
detail, see fig. S6C. Data are representative of
biological triplicate experiments. (F) Quantitation
of SRY in NR5A1+cells in E11.5 gonads of the
indicated genotypes. Data are shown as means ± SD.
Numbers of analyzed cells are shown in parentheses
above each bar. ***P< 0.001 between the indicated
groups, ANOVA with Tukey’s post hoc test.

**(P = 0.009)

B

WT (XY) Sry-T-Nr5a1R26-CreKI/+ (XX); Sry-SNr5a1-R26-CreKI/+ (XX); Sry-SNr5a1-R26-CreKI/KI(XX);

A

Sry-S

-R26

KI/+

Sry-T (XX)

-R26

KI/+

(XX)

Nr5a1-Cre

FLAG

(kDa)

116

66

66

40

`-ACTIN

--++

0.0 3.81 0.51

0.5

1.0

1.5

2.0

Relative expression of

Sry ns (P = 0.100)

Sry-T

-R26

KI/+

(XX)Sry-S

-R26

KI/+

(XX)

C

D

Sry-S-R26KI

Sry-T-R26KI CAG pA 3xFlagSry-TpA

Rosa26 locus

(WT)

Cre mediated exision

CAG pA 3xFlagSry-SpA

EF

***

***

Nr5a1-Cre --++

loxPloxP

***

SryTR26 (XX) SrySSR26 (XX) SrySSR26 (XX)

***

SRY

SRY

/NR5A1

/DAPI

WT (XY) Sry-TNr5a1-R26-KI/+ Cre(XX); Sry-S-Nr5a1R26-CreKI/+ (XX); Sry-S-Nr5a1R26-CreKI/KI (XX); WT (XX)

50 μm

0

50

100

Nr5a1-Cre

;

Sry-T

-R26

WT (XY)KI/+ (XX) WT (XX)

Nr5a1

• Cre

;

Sry-S

-R26

KI/+ (XX)

Nr5a1

• Cre

;

Sry-S

-R26

KI/KI (XX)

SRY intensity

in NR5A1 positive cells (A.U.)

(95)

(107)

(90)

(65)

(63)

A C WT (XY) Sry exon2^6 (XY) WT (XX)

XY HSP-Sry (+)

XY Sry HSPexon2-Sry 6 (+)

XX

F0 XX

Deletion of by CRISPR/Cas9Sry exon2

Sry exon2 (^6) Phenotype analysis
F1
D E
poly Q Exon2 6
Exon1
HMG
Sry exon2 6
WT
poly Q
Exon1 Exon2
HMG
SRY
SRY
/NR5A1
/DAPI
WT (XY) Sry exon2 6 (XY) WT (XX)
B
WT (XY) WT (XX)
Sry
exon2
6 (XY)
0
50
100 ***
SRY intensity
in NR5A1 positive cells (A.U.)
XY HSP-Sry (-) 50 μm

• Genome
  editing
  (74)
  (81)
  (61)
  Tes
  Ova Ova
  Fig. 2.Sry-Tis necessary for male development in mice.(A) Deletion ofSry
  exon2 by CRISPR/Cas9. (B) Establishment ofSry-T-deficient mice. Procedures are
  detailed in fig. S5, A and B. To circumvent potential male infertility ofSry-T–deficient
  male mice, we used XY zygotes carrying theHSP-Srytransgene ( 17 ) for genome
  editing. The resultant F 1 generation without theHSP-Srytransgene was used for
  phenotypic analysis. (C)Sry-T–deficient mice exhibited male-to-female sex reversal
  (n> 40). External genitalia, nipples (top panels), and internal genitalia (bottom
  panels) of adult mice are shown. Tes, testis; Ova, ovary. (D) Coimmunostaining
  profiles of SRY and the gonadal somatic marker NR5A1 in E11.5 gonads of the
  indicated genotypes. Data are representative of biological triplicate experiments.
  (E) Quantitation of SRY in NR5A1+cells in E11.5 gonads of the indicated genotypes.
  A.U., arbitrary units. Data are shown as means ± SD. Numbers of analyzed cells
  are shown in parentheses above each bar. ***P< 0.001 between the indicated
  groups, one-way analysis of variance (ANOVA) with Tukey’s post hoc test.
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